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Robinson Flashcards

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1
Q

Purpose of endosomes

A

Sorting of endocytosed material

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2
Q

Protein import into the ER (ER destined)

A

N-terminal signal sequence, 15-20 AAs long
SRP binds to sequence on growing polypeptide
SRP binds to SRP receptor near translocation channel
SRP leaves, growing chain pushes through channel
Signal sequence left in membrane, cleaved by signal peptidase

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3
Q

Structure of SRP

A

Ribonucleoprotein
7S RNA and various proteins
Binding of signal sequence is modulated by NAC
Elongation is stopped until SRP docks

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4
Q

The Sec61 channel structure

A

Used for import to ER
Hetero trimeric complex of aby subunits
Also used for post translational with Hsp70/Hsp40

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5
Q

Embedding proteins into the ER

A

Sequence of hydrophobic stop transfer

Anchors to membrane

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6
Q

Function of the ER

A

Protein folding- PDI rearranges disulphide bonds. Chaperones can bind to hydrophobic regions
Glycosylation
Quality control

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7
Q

Glycosylation of proteins

A

Lipid linked oligosaccharide
Asn-X-Ser/Thr
Anchored to membrane by -P- to dolichol
Cleaved from membrane to join to Asn by transferase

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8
Q

ER quality control-Protein folding

A

N-glycosylation increases the solubility of unstructured chains. Chaperones and folding factors are recruited when time for folding expired

Misfolded proteins remain bound to chaperones and are degraded

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9
Q

ER quality control- Glucose trimming

A

Terminal glucoses are removed by a-glucosidases to form the Glc1Man9GlcNAc2 sugar
Glucosidase I and II remove 2 outermost residues belonging to N-glycans branch A
Chaperones bind to exposed hydrophobic regions and prevent vesicles

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10
Q

Chaperone proteins

A

Receptors IRE1 and PERK are activated by oligermerisation and trans phosphorylation when misfolded proteins bind
The Bip inhibitor leaves the receptor

IRE1 cleaves XBP1 mRNA to activate it
IRE1 also forms RIDD -> mRNA attached to ribosome degradation

PERK phosphorylates eIF2 to inhibit translation
ATF4 (mRNA) escapes block and creates -> CHOP + GADD34
These are factors for amino acid metabolism

When bip is released, activates ATF6 receptor
ATF6 Trans located to golgi -> luminal domain cleaved
Moves to nucleus and expresses molecules for chaperone pathways

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11
Q

Purpose of ER

A

Lipid synthesis

Secretory pathway

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12
Q

Principles of mitochondrial import

A 
Imported post translationally but unfolded
Made with transient n terminal extension
Internal targeting signals not removed
Trans located through TOM
Presequence directed to TIM23
Inner membrane -> TIM22
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13
Q

TIM23

A

Presequence containing proteins
Example of sequence- COXIV sequence is an Amphipathic helix
Transport depends on Ψ and mtHsp70(ATPase)

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14
Q

Mitochondrial targeting sequences

A

Positive hydrophobic residues
Amphipathic helices, used to bind to receptor in outer membrane
Positive charge required for Ψ driven transport
Cleaved matrix proteins have an Arg at -2 or -3
Processing matrix by MPP

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15
Q

Import of COXIV-DHFR with methotrexate

A

Methotrexate- protein only processed by MPP
Must have 50+ residues between folded DHFR and processing site
Tim23 and TOM are stacked at contact sites where 2 membranes are close enough for translocation

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16
Q

Structure of TOM complex

A

Tom 20 -> 22 -> TOM40

Tom80 -> TOM40

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17
Q

PAM complex

A 
PAM 1618
Tim44
mtHsp70
Mge1
This acts as an ATP powered pulling mechanism
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18
Q

Brownian ratchet model

A

Protein must be unfolded
Brownian movement only causes fluctuation across membrane
Protein trapped so cannot move back
Dragged through, unwinding

Protein targeting needs ATP
translocation needs ATP and electrical gradient

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19
Q

Inner and outer membrane protein structure

A

Inner are a helical

Out are b barrel

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20
Q

Stop transfer mechanism

A

Proteins arrested in membrane during translocation
E.g. CoxVA in inner membrane
Cleavage by matrix protease
Stop transfer sequence

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21
Q

Conservative sorting

A 
Protein is imported into matrix
Instead by ancestral pathway 
Example- ATP synthase unit 9 
Cleavage by matrix protease
Internal sequences recognised by Oxa1
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22
Q

What determines the route of an inner membrane protein

A

Proteins without homologue use stop transfer (TIM proteins)
Proteins with bacterial homologues use conservative sorting
Could be due to sequence and charges around hydrophobic domain

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23
Q

TIM22

A

Similar to TIM23
Import of AAC (an ADP ATP carrier) and phosphate carrier (PiC)
In the preprotein Sequences are recognised by Tom70 and Tim22
Chaperone through tom70 and into general import pore linked to tim9/10
This transports it to tim22/54

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24
Q

Targeting signals of chloroplast proteins

A

Hydroxylated amino acids, 30-100 residues
May be phosphorylated at Ser or Thr
Transit peptides bind targeting factor in cytosol
Assisted by cytosolic chaperones
Recognised by TOC
Presequences removed by stromal processing peptidase
Thylakoid have additional targeting signal

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25
Recognition of preprotein by TOC
Preprotein -> GTPase domain of Toc159 Complex docks at outer membrane via interaction with toc159 and toc34 Toc159/34 interaction -> GTP hydrolysis -> integration of toc159 and insertion of preprotein After translocation toc139 and 34 exchanges GDP for GTP Dissociation of toc159
26
Translocation by the TOC complex
Transit peptide binds to toc34 Stimulates GTP hydrolysis by toc34 which transfers preprotein to toc159 GTP hydrolysis by toc159 causes conformational change forcing the preprotein through the translocation channel More rounds of GTP hydrolysis completes process
27
3 stages of chloroplast envelope translocation
Transit peptide makes reversible contacts with receptors of the TOC complex Preprotein inserts into the TOC complex and makes contact with TIC components (low ATP conc) Preprotein is trans located into Stroma and the TP is cleaved by SPP (high ATP needed)
28
Import of proteins into the thylakoid lumen
Made with bipartite targeting signal One for transport across envelope, one for transport into thylakoid Lumenal proteins follow Sec or Tat pathway Depends on signal peptide Transport is post translational
29
Sec pathway
Transported unfolded Depends on ATP and stromal proteins or cytoplasmic proteins SPase removes the targeting signal at the trans side of the membrane After translocation, proteins fold to active conformation
30
Tat pathway
Transported folded Relies solely on proton motive force SPase removes the targeting signal at the trans side of the membrane Conserved residues in targeting signals
31
Protein export in bacteria
Putative RR signal peptide Consensus sequence S/TRRxFLK Tat substrates in e.coli bind cofactors and are involved in respiration Cofactor insertion takes place in the cytoplasm, why folded proteins are trans located
32
Possible mechanism for the tat system
Two seperate complexes are present TatA + tatABC complex Only tatBC in thylakoids Substrate targeted to the membrane and binds to tatBC tatA is then recruited to the core complex to make a pore of the right size The protein is trans located TatA dissociates to close the pore
33
Why transport proteins to the nucleus
Nuclear function proteins are synthesised in the cytoplasm Nuclear envelope breaks down in mitosis Nuclear and cytoplasm mix Components must be resorted after cell division
34
Nuclear localisation signal + gated transport
Pro-pro-Lys-Lys-Lys-Arg This needs to be on surface Recognised by nuclear import receptors These bind to nuclear pore fibrils extending into cytosol Protein and receptor enter nucleus Import receptor recycled Gated transport- larger than 60kDa need ATP
35
Mechanism of nuclear pore transport
Nuclear localisation signal associates with import receptors These bind the nuclear pore complex (act as chaperone) Complex moves through pore (unknown) Once inside, Ran-GTP displaces import receptor Ran-GTP receptor complex travels out of nucleus GTP hydrolysis and receptor released in cytoplasm
36
Monomeric GTPases
Cell has high GTP, so only anti-clockwise GTP -> GDP by GAP removing Pi Hydrolysis energy used to power cellular functions Reverse reactions stimulated by GEF
37
Ran-GAP/GEF in nuclear transport
Ran-GAP -> lots of Ran-GDP in cytosol In the nucleus there is lots of Ran-GEF therefore Ran-GTP High ran-GTP promotes dissociation of NLS from import receptor Most nucleus export is RNA
38
Export of RNA from nucleus
Forms complex of ribonucleoprotein The protein component contains a NES that is recognised by export proteins mRNA is bound by hnRNP after full splicing (ensures mature)
39
Modification of oligosaccharide added in ER
Enzymes in cisternae can add and remove sugar residues | Also phosphorylation of mannose residues
40
Role of protein glycosylation
Inhibition does not affect secretion, may affect folding Oligosaccharides can make protein resistant to protease digestion Also function in cell to cell adhesion Connected via Asn
41
Specific proteolytic cleavage
Some proteins are synthesised as larger precursor proteins and then cleaved by TGN molecules to activate Some proteins entering Golgi need to return to ER, ER retention signal recognised by cis golgi
42
ER -> Golgi transport
Proteins bud off in vesicles and form a vesiculotubular cluster (vesicles with vesicles inside) This travels along micro tubules to the Golgi It then breaks down into ER-Golgi intermediate component Assembly of new cis-Golgi cisternae from VTC elements
43
Protein sorting in the trans Golgi network | 4 destinations
4 destinations- stay, lysosomes, constitutive or regulated secretion
44
Lysosomes and trans Golgi network
Acidic ph due to proton pump Lysosomal enzymes and membrane proteins synthesised in ER Mannose residue is phosphorylated at Golgi Mannose-6-phosphate recognised by vesicle coat proteins Clathrin coat bound to adaptin and directed to lysosomes
45
Sorting receptor for lysosomes
Cargo binds to sorting receptor Sorting receptor binds to adaptin which binds to clathrin to make coat Vesicle buds off to lysosomes
46
Vesicle budding
Clathrin molecules shape vesicle Dynamin assembles round neck of vesicle Hydrolases GTP contracting the ring and vesicle leaves
47
Structure of clathrin
Triskelion shape Each leg has heavy and light chain These polymerise to form a polygonal lattice with an intrinsic curvature
48
COP I coated vesicles (Golgi -> ER)
Requires ARF to release GDP and bind GTP complex then binds to ARF receptors on Golgi COP I coatamers bind to ARF and other proteins inducing budding
49
COP II coated vesicles (ER -> Golgi)
GEF in donor membrane interacts with GTPase Sar1 -> exchange Sar1-GTP extends fatty acid tail that extends into membrane COPII assembles on Sar1 to form vesicle
50
Vesicle fusion
Uncoating exposes vSNARE ,recognised by tSNARE/SNAP25 complex NSF, a/b/gSNAP proteins bind to stabilise energy barrier Rab-GTP hydrolysed, causes fusion and releases complex Vesicle with vSNARES returns to donor membrane
51
Conformational changes in vSNARE tSNARE complex
Stalk formation Hemi fusion Fusion This expels water, lipid contacts involve syntaxin, synaptobrevin and SNAP25
52
Separation of the vSNARE/tSNARE complex
NSF cycles between the membrane and cytosol catalysing disassembly Uses ATP to unravel interactions Stops tSNARES from always being active Ran monitors NSF
53
Constitutive pathway
Bulk flow All proteins except for ER and Golgi retention, transport to lysosomes or secretory vesicles Plasma membrane proteins and lipids etc. Some secretion to blood e.g. Albumin
54
Regulated pathway
Selective aggregation in trans Golgi Secondary structure important Uptake of aggregate into immature secretory vesicles
55
Endocytosis pathways
Pinocytosis- fluid and molecules, all cells | Phagocytosis- large molecules, specialised cells e.g.macrophage
56
Fates for proteins after endocytosis
Recycling- receptors to same place on membrane Transcytosis- different place on membrane Degradation- take to lysosomes
57
Serine proteases
Chymotrypsin- likes aromatic side chain on residue whos carbon is being cleaved Trypsin- prefers a positively charged Lys or Arg residue There is Nucleophillic attack of the hydroxyl O of a Ser on the carbonyl carbon Forms an acyl enzyme intermediate
58
Aspartate proteases
Pepsin, Remin, HIV-protease One Asp accepts a proton from an active site H2O which attack the carbonyl carbon of the peptide linkage The other -/0 donates a proton to the oxygen of the peptide carbonyl group at the same time
59
Zinc proteases
Carboxypeptidases, matrix metalloproteases, one lysosomal protease Some involved in degradation of EM during tissue remodelling (collagen) Some have roles in cell signalling, can release cytokines or growth factors by cleave of membrane preproteins
60
Cystein proteases
Also known as Capthespsins De protonation of the Cys SH by adjacent His residue Followed by Nucleophillic attack of the cysteine S on peptide carbonyl carbon Thioester linking the new carboxyterminus to cysteine thiol is an intermediate of the reaction
61
Protein turnover
N-end rule, half life correlates to N terminal residue Phe, Leu, Asp have less than 3 mins PEST proteins very fast
62
Joining of ubiquitin to protein
Terminal carboxyl of ubiquitin joined to Cys on E1 (ATP dependent) Ubiquitin transferred to a S group of E2 E3 transfers activated ubiquitin to e amino group of Lys of protein recognised by E3 More ubiquitins are added to form chain Linked by Lys residues 4+ targets proteins for degradations
63
Destruction box sequence
Recognised by a domain of the corresponding ubiquitin ligase | Present in mitotic cyclins etc.
64
Ubiquitin ligases (E3)
Some have HECT domain containing a Cys residue that participates in ubiquitin transfer Some have a RING finger where Cys and His residues are ligands to 2Zn2+ ions
65
The proteasome core complex
20S sedimentation coefficient 7 a type proteins form 2 a rings at the ends 7 b type proteins form the 2 central b rings Cavity with 3 joined compartments Proteases activities are associated with 3 of the b subunits
66
The 3 b protease subunits
Chymotrypsin like activity- preference for Tyr or Phe at P1 Trypsin like activity- Arg or Lys at P1 Post-glutamyl activity- Glu or acidic residue at P1 Unique family of threonine proteases, conserved Thr involved at catalysis at each active site. Pre proteins activated by cleavage -> taken to lumenal surface
67
The 19S cap
Unfolds ubiquitin proteins, removes ubiquitn and provides passage Outermost lid is ring of 8 proteins Innermost base is 6 ATPases - unfolding chaperones Isopeptidases disassemble ubiquitin and recycle 19S + 20S -> 26S proteasome
68
The 11S cap
Regulatory cap- heptameric complex of PA28 Allows non ubiquitin proteins and peptides to pass through Binding alters conformation of core complex a units opening gate

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